Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen that poses a significant public health threat in the context of nosocomial infections, particularly for immunocompromised patients such as burn victims, cancer patients, and individuals having cystic fibrosis or AIDS. P. aeruginosa is also prone to antibiotic resistance, through both intrinsic and acquired mechanisms. Thus, there is a great need for the development of novel anti-Pseudomonas drugs that address unexploited targets, and this is a specific focus of this RFA. To address this problem, we propose herein to develop novel small molecule antibacterials that target the P. aeruginosa quinolone (Pqs) quorum sensing system. This is a pharmacologically validated target in a mouse model of infection and is distinct from acyl homoserine lactone (Las, Rhl) quorum sensing systems. Quinolones are small molecules that are biosynthesized by the bacteria and used in cell-cell signaling. They control expression of a variety of bacterial virulence factor genes that are associated with pathogenicity but are not required for bacterial viability or growth. As such, novel antibacterials that target such virulenc factors are thought less likely to elicit drug resistance compared to traditional bacteriotoxic and bacteriostatic antibiotics. Building upon extensive previous work from the three participating laboratories (Tan, Rahme, Pesci), we will use mechanism- and structure-based rational drug design to develop small molecule inhibitors of PqsA, an anthraniloyl-CoA synthetase that catalyzes an essential step in P. aeruginosa quinolone biosynthesis. PqsA has been validated as an effective antibacterial target in a mouse model using simple substrate analogues, but more potent and specific inhibitors are required to exploit fully the therapeutic potential of this target. In the R21 phase, we will synthesize first-generation inhibitors using a rational design strategy that has been applied successfully to related targets in the Tan lab, then evaluate their activities in biochemical and cellular assays for PqsA activity and quinolone production established previously in the Pesci and Rahme labs. In the R33 phase, we will optimize the biochemical, cellular, and pharmacological properties of the inhibitors to develop lead compounds that will then be advanced to in vivo evaluation in established mouse models of P. aeruginosa infection in the Rahme lab. This multidisciplinary collaboration comprises the necessary combined expertise in synthetic organic chemistry, medicinal chemistry, biochemistry, pharmacology, and microbiology. Our long- term goals are to develop one or more advanced candidates for further preclinical and clinical evaluation as novel antibiotics to combat P. aeruginosa and potentially other pathogenic Gram-negative bacteria.